Molecular Studies Of Familial Vesicoureteral Reflux

Vesicoureteral reflux(VUR) is a congenital urinary tract defect caused by abnormal insertion of the ureter within the bladder wall.This malformation leads to a defective ureterovesical junction in which urine flows retrogradely from the bladder to the ureters and kidneys especially during micturiation.VUR affects approximately 1%of the population and is associated with recurrent urinary tract infections,renal malformations,and renal scarring defined as reflux nephrology(RN).Reflux nephrology is the most common cause of the end-stage renal disease in children and adolensents.VUR can be classified intoâ… -â…¤grades.VUR patients who belong toâ…£andâ…¤grade need surgical theraphy.The voiding cystourethrography(VUCG) is the gold standard diagnostic methods and must include the voiding phase.It is not feasible to screen the whole population for reflux.The hereditary and familial nature of vesicoureteral reflux is now well recognized.To screen the candidate genes for vesicoureteral reflux is helpful for early diagnosis and the pathogenesis of VUR will be better understood by clarifying the molecular background.Familial clustering of VUR implies that genetic factors may have an important role in the pathogenesis of reflux.The concordance is higher in monozygotic twins than in dizygotic ones(80-100%and 35-50%,respectively).The risk for children if one parent had been diagnosed with VUR is reported to be 50-66%.The model of inheritance for VUR is multifactorial or autosomal dominant with reduced penetrance. Three genome-wide linkage studies reported to date with linkage to chromosome 1p13,chromosome 2q37 and chromosome 3q22.2-23,respectively. During the development of the embryo,the metanephric mesenchyme induces the nearby Wolffian duct to evaginate an epithelial tube,the ureteric bud.This invades the metanephric mesenchyme,which in response condenses,proliferates,and undergoes an epithelial transformation.The proximal end of the ureteric bud remains attached to the mesonephric duct and elongates,becoming the distal ureter,ureter-vesico junction and the bladder trigone.The ureteric bud position on the Wolffian duct to the nephrogenic blastema can affect the orifice in the bladder and urethra and the normal structure of the vesico-ureteric junction.Many genes involved in ureteric bud induction.Such as:Glial cell line-derived neurotrophic factor(GDNF),Paired box gene2(PAX-2),Rearranged during transfection protooncogene(RET).Recent studies in mice have shown that gdnf is expressed along the Wolffian duct during embryological development and that the amount of gdnf is determined by slit2 and its receptor robo2 thereby affecting the outgrowth of the ureteric bud. Homozygous deletion of either robo2 and slit2 in the mouse results in supemumberary ureteric buds that form multiple kidneys and ureters.Interestingly one patient with isolated VUR was described with a translocation between the Y-chromosome and chromosome 3 with a break point between exon 1 and 2 of the ROBO2 gene.Two novel ROBO2 missense variants were found in two unrelated families by screening on in 124 families with VUR or congenital abnormalities of kidney/urinary tract(CAKUT).Another study on 95 unrelated cases has identified a relatively high frequency of ROBO2 variants(5.1%) in familial cases of VUR or VUR/CAKUT.RET is expressed by the mesonephric duct,the ureteric bud,and the future bladder. The RET-/- mice have ureteral abnormalities such that the distal ureters are not connected to the bladder but instead remain attached to sex ducts.Overexpression of RET leads to vesicoureteric reflux in mice.RET Gly691Ser mutation is associated with primary vesicoureteral reflux in the French-Canadian population from Quebec. In summary,ROBO2,SLIT2 and RET are all candidate genes for familial vesico-ureteric reflux.We collected 54 families with reflux from Gothenburg, Sweeden.Every family has at least 2 person with vesico-ureteral reflux.Then overall aim of this thesis is to study these candidate genes in familial vesico-ureteric reflux. This thesis includes two parts:the first part is to further study the ROBO2 and SLIT2 genes on vesico-ureteric reflux by sequencing on 54 index patients.And do assiociation studies and function prediction.The second part is to study the RET polymorphism rs1799939:G>A and familial vesico-ureteral reflux by genotyping on 54 index patients and their relatives and 163 controls.Part one:Study the roles of ROBO2 and SLIT2 genes in familial vesieo-ureteral refluxObjective:This study the mechanism of ROBO2 and SLIT2 genes in familial vesico-ureteral reflux by mutation screening on 54 unrelated patients with primary VUR from Sweden.Material:Fifty-four families with more than one affected relative with non-syndromic vesico-ureteral reflux were identified through medical records in Sweden.One affected from each family was selected for mutation screening.As control group for selected polymorphisms,we used DNA from an anonymous sample constituted by 96 healthy voluntary blood donors at Karolinska University Hospital, Stockholm.In families with 2 affected,altogether in 17 families sibs were affected.In 16 families VUR was inherited from a parent and in our study in 14 cases of 16 it was maternally inherited.The grade of VUR in the probands was gradeâ… (n=1),â…¡(n=5),â…¢(n=12),â…£(n=17),andâ…¤(n=8) degree vesico-ureteral reflux.In the remaining 11 cases,the severity of vesico-ureteral reflux was not available.7 of the probands have duplex collecting systerm,2 have pyeloureteral junction stegnosis,1 has duplex collecting systerm and ureterocele bilateral.Methods:(1) DNA was extracted from EDTA preserved blood,and isolated according to standard procedures.(2) Exon-flanking primers(exon 1-26 in ROBO2 gene,exon 1-37 in SLIT2 gene) were designed using Primer 3 program.PCR reactions were performed with AmpliTaq Gold and DyNAzyme^TM EXT DNA Polymerase in a final volume of 25 ul.(3) After ExoSap-IT enzyme treatment,the PCR fragments were sequenced on both direction using BigDye(?) Terminator v3.1 kit and analyzed in ABI Prism 3730 Sequencer.Sequence analysis was preformed with the program SeqScape v2.5.(4) HomoloGene detected putative homologs of human SLIT2 sequences.Splicing prediction was performed with four computer programs:BGDP Splice Site Prediction,NetGene2,GeneSplicer,Human Splicing Finder.Secondary structures were predicted with NNPREDICT.The program Polyphen was used to predict the possible impact of an amino acid substitution on the structure and function of ROBO2 and SLIT2 Proteins.Results.Exons 1-26 of the ROBO2 gene were normal in all of the 54 cases.Six sequence variants were observed in the exon-intron boundary area.Two of them: IVS13+43C>T,IVS21-40T>C have not been described before.The sequence variant IVS13+43C>T was found in heterozygous in one patient and not found in 95 control DNA samples.It may represent a private mutation.The sequence variant IVS21-40T>C was found in two patients respectively in heterozygous and homozygous form.One of 95 control DNA samples presented this variant in heterozygous form.Four patients presented the heterozygous sequence variant IVS3-3 (allele frequency 3.6%) which also found by Bertoli-Avella.This sequence variant did not co-segregate with VUR in four families.Two of the 95 control DNA samples presented this variant in heterozygous form and it may thus represent a single-nucleotide polymorphism(minor allele frequency 1%).Three SNPs:rs5850333, rs17525412,rs3821735 were also found in the patients.All of them didn't co-segregate with VUR.None of the above sequence variants was predicted to alter gene splicing.Several single-nucleotide variations were found in the SLIT2 gene.The variants c.4253C>T/p.Ala1418Val,c.1209A>T,c.2211C>T(rs7690492),c.2550A>C (rs17542486) are all within the exons,and 3'UTR*51A>G(rs1379659) in the 3'UTR. The variant c.4253C>T(p.Ala1418Val) is the only non-synonymous sequence variant found.It converts the amino acid 1418 from alanine to valine,both being hydrophobic amino acids.The variant was found in heterozygous state in one male and one female (from family 50 and 53).The boy's variant was inherited from the father,whereas his mother had a normal sequence.His sister with VUR had the same sequence variant, however his affected brother and aunt did not.In family 50,only the affected aunt had the sequence alteration,the proband and his nephew didn't have.This missence variant changed the secondary structure of the SLIT2 protein,but may have little effect on the three-dimension structure and function.The sequence changes c.1209A>Y,c.2211C>T(rs7690492) and c.2550A>C (rs17542486) are all synonymous.The site 1209bp was described as a SNP (rs34224151,A>G) before but we found a heterozygous variant A>T on this site in one patient.None of the 95 control DNA samples presented this variant and it may thus represent a private mutation.Other gene variants:rs61790829,rs16869654, rs2290752,IVS7+81C>G(not described before),rs4443280,rs543593,rs519813, rs55972345,rs10428394,rs55798019,rs2290750,rs12506323,rs2290751,rs3733510, rs35891001 were found in the exon-intron boundary area of the SLIT2 gene.The new gene variant IVS7+81C>G were found in heterozygous in 8 cases and in homozygous in 3 cases.It didn't segregate with VUR in families.18 of 95 control DNA samples presented this variant in heterozygous form.3 presented in homozygous form.The minor allele frequency is 0.123.It may thus represent a single-nucleotide polymorphism.None of these sequence variants was predicted to cause any splicing alteration or protein structure modifications by the previously mentioned methods.Conclusion:In summary,gene variants in ROBO2 and SLIT2 are rare causes for VUR. To our knowledge this study demonstrates for the first time the SLIT2 gene missense variant in familial vesico-ureteral reflux patients,however,functional studies and validation in other cohorts are warranted.The reduced sample size probably limited the power of our study for finding novel pathogenic variants.Neverthe-less,our study populations consisted of familial cases,and this design was aimed at increasing the chance of finding genetic causes of VUR.In summary,gene variants in ROBO2 and SLIT2 genes are rare causes of VUR in humans,providing further evidence for the genetic hetero- geneity of this disorder. Part two:Study if the RET polymorphism rs1799939.G>A associate with familial Vesicoureteral refluxObjective:To investigate the association between the RET polymorphism rs1799939: G>A and familial vesico-ureteric reflux in swedish population.Material and methods:Fifty-four families with more than one affected relative with non-syndromic vesico-ureteral reflux were identified through medical records in Sweden.We collected 54 index patients and their relatives' DNA and 163 control's DNA.We use Teqman genotyping method to genotype the RET polymorphism rs1799939:G>A.Results:35 of the index vesico-ureteral reflux patients have the GG genotype,15 have the GA genotype.2 have the AA genotype.107 of the control DNA have the GG genotype.48 have the GA genotype.8 have the AA genotype.By X² test,there was no significant difference between the cases and controls on A allele frequency.The RET polymorphism rs 1799939:G>A co-segeregated with VUR in 5 families.Conclusion:The RET polymorphism rs 1799939:G>A(Gly691 Ser) could generate a spectrum of deficiencies of ureteral development in some VUR patients.The cause of VUR can vary in different populations.The reduced sample size is probably limiting the power of our study in finding association between the RET polymorphism rs 1799939 and VUR.In summary,our results giving further evidence for the genetic heterogeneity of this disorder.